LOCUS       sequence-357026-        7661 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression plasmid for SARS-CoV-2 nsp4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7661)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7661)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pGBW-m4134223
FEATURES             Location/Qualifiers
     source          1..7661
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1508..1528
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1529..1570
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     polyA_signal    1623..1744
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1634..1653)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1688..1707
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1859..1905
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1935..2071
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2116..2187
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2413..3001)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2493..2512)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3501..4710
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4547..4566
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4714..5313
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4714..4733)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5210..5230
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5340..5388
                     /note="synthetic polyadenylation signal"
     CDS             join(5654..5722,5723..6514)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5872..5891)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6527..6663
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6815..6886
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6891..6937)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6955..7003
                     /note="synthetic polyadenylation signal"
     enhancer        7084..7387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7389..7591
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7542..7562
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 caccatgaag attgtaaata actggttgaa acaactaata aaggttactt tagtgttcct
       61 gtttgtcgcc gcaatcttct atcttatcac cccggtgcac gtgatgagta aacatacaga
      121 cttttcttca gaaattatcg ggtacaaggc tatcgatggc ggtgtaacgc gcgacattgc
      181 ctccacagat acctgcttcg cgaacaaaca cgctgacttt gatacttggt tcagccagcg
      241 aggaggctcg tacaccaatg acaaagcctg tccactgatc gcagccgtga taactagaga
      301 ggttggattc gtcgtccctg ggctccccgg gacaattctc cgtacaacca acggcgactt
      361 tctgcatttt ctgccccggg tgttctccgc agtcggtaat atctgttata ctccatctaa
      421 gctcattgag tacacggatt ttgctaccag cgcgtgcgtg ttggccgcag aatgcaccat
      481 atttaaggac gcttctggaa aacctgtgcc ctattgttac gataccaatg tgcttgaggg
      541 ctcagttgcc tatgaaagtc tgaggccaga cactagatac gttttaatgg atggaagcat
      601 catccagttc ccaaacacgt atctggaggg gtccgtgcgg gtcgtgacca cattcgattc
      661 agaatactgc cgccacggta catgcgagag gtctgaggct ggcgtatgtg tgtcaactag
      721 cggtcgatgg gttctaaata acgattatta tcgttctctg cccggcgttt tttgtggagt
      781 ggacgccgtt aaccttttga ccaacatgtt tacacctctc attcagccca ttggtgcact
      841 ggatatctcc gcgagtattg tggctggggg aatagtcgcc attgtcgtaa cctgcttggc
      901 atactatttc atgcgtttca ggcgagcttt tggcgaatac tcacatgtcg tggccttcaa
      961 tactcttctg tttctgatga gcttcacggt actctgtctg accccagtat acagtttcct
     1021 acctggggtg tattctgtga tctacttata cctcacattt tacctgacca atgacgtgtc
     1081 ctttctggcc cacatccaat ggatggttat gttcacgccg ctcgtgcctt tctggattac
     1141 aatcgcttat ataatctgta ttagcacgaa gcatttctat tggttctttt ccaactactt
     1201 aaagcggcgc gtcgtattta atggggtatc gtttagcact tttgaggaag ccgcgctctg
     1261 cactttcttg cttaataagg agatgtattt gaaattaaga tctgacgttc tgcttccttt
     1321 aacacagtac aacagatacc tggcgttgta taacaaatac aaatatttct cgggagccat
     1381 ggatacaact tcctatagag aggctgcttg ttgccacctt gcaaaggcac tcaacgactt
     1441 cagtaacagc ggcagtgatg tgctttatca gccccctcag acttcaataa catccgccgt
     1501 cctccaagaa aacctttact ttcagtcggg gaaacctata cccaatccac ttctcggctt
     1561 agattctacc taagctagct tgactgactg agatacagcg taccttcagc tcacagacat
     1621 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     1681 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     1741 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt
     1801 tttttaaagc aagtaaaacc tctacaaatg tggtattggc ccatctctat cggtatcgta
     1861 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgtgccc ctcgggccgg
     1921 attgctatct accggcattg gcgcagaaaa aaatgcctga tgcgacgctg cgcgtcttat
     1981 actcccacat atgccagatt cagcaacgga tacggcttcc ccaacttgcc cacttccata
     2041 cgtgtcctcc ttaccagaaa tttatcctta aggtcgtcag ctatcctgca ggcgatctct
     2101 cgatttcgat caagacattc ctttaatggt cttttctgga caccactagg ggtcagaagt
     2161 agttcatcaa actttcttcc ctccctaatc tcattggtta ccttgggcta tcgaaactta
     2221 attaaccagt caagtcagct acttggcgag atcgacttgt ctgggtttcg actacgctca
     2281 gaattgcgtc agtcaagttc gatctggtcc ttgctattgc acccgttctc cgattacgag
     2341 tttcatttaa atcatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     2401 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     2461 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     2521 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     2581 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     2641 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     2701 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     2761 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     2821 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     2881 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     2941 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     3001 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     3061 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     3121 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     3181 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcatt
     3241 taaatttccg aactctccaa ggccctcgtc ggaaaatctt caaacctttc gtccgatcca
     3301 tcttgcaggc tacctctcga acgaactatc gcaagtctct tggccggcct tgcgccttgg
     3361 ctattgcttg gcagcgccta tcgccaggta ttactccaat cccgaatatc cgagatcggg
     3421 atcacccgag agaagttcaa cctacatcct caatcccgat ctatccgaga tccgaggaat
     3481 atcgaaatcg gggcgcgcct ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc
     3541 cctcgtcacg gcgagcgctg ccacgtcaga cgaagggcgc aggagcgtcc tgatccttcc
     3601 gcccggacgc tcaggacagc ggcccgctgc tcataagact cggccttaga accccagtat
     3661 cagcagaagg acattttagg acgggacttg ggtgactcta gggcactggt tttctttcca
     3721 gagagcggaa caggcgagga aaagtagtcc cttctcggcg attctgcgga gggatctccg
     3781 tggggcggtg aacgccgatg attatataag gacgcgccgg gtgtggcaca gctagttccg
     3841 tcgcagccgg gatttgggtc gcggttcttg tttgtggatc gctgtgatcg tcacttggtg
     3901 agtagcgggc tgctgggctg gccggggctt tcgtggccgc cgggccgctc ggtgggacgg
     3961 aagcgtgtgg agagaccgcc aagggctgta gtctgggtcc gcgagcaagg ttgccctgaa
     4021 ctgggggttg gggggagcgc agcaaaatgg cggctgttcc cgagtcttga atggaagacg
     4081 cttgtgaggc gggctgtgag gtcgttgaaa caaggtgggg ggcatggtgg gcggcaagaa
     4141 cccaaggtct tgagcccttc gctaatgcgg gaaagctctt attcgggtga gatgggctgg
     4201 gcaccatctg gggaccctga cgtgaagttt gtcactgact ggagaactcg gtttgtcgtc
     4261 tgttgcgggg gcggcagtta tggcggtgcc gttgggcagt gcacccgtac ctttgggagc
     4321 gcgcgccctc gtcgtgtcgt gacgtcaccc gttctgttgg cttataatgc agggtggggc
     4381 cacctgccgg taggtgtgcg gtaggctttt ctccgtcgca ggacgcaggg ttcgggccta
     4441 gggtaggctc tcctgaatcg acaggcgccg gacctctggt gaggggaggg ataagtgagg
     4501 cgtcagtttc tttggtcggt tttatgtacc tatcttctta agtagctgaa gctccggttt
     4561 tgaactatgc gctcggggtt ggcgagtgtg ttttgtgaag ttttttaggc accttttgaa
     4621 atgtaatcat ttgggtcaat atgtaatttt cagtgttaga cttgtaaatt gtccgctaaa
     4681 ttctggccgt ttttggcttt tttgttagac aacatgaccg agtacaagcc cacggtgcgc
     4741 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     4801 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     4861 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     4921 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     4981 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     5041 gagggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     5101 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     5161 gccgccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     5221 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     5281 acctggtgca tgacccgcaa gcccggtgcc taggctagta tgtaagccta gtcttagata
     5341 ataaaatcgc tatccatcga agatggatgt gtgttggttt tttgtgtgtg taacgctagg
     5401 cgcgcctggt gtaccgagaa cgatcctctc agtgcgagtc tcgacgatcc atatcgttgc
     5461 ttggcagtca gccagtcgga atccagcttg ggacccagga agtccaatcg tcagatattg
     5521 tactcaagcc tggtcacggc agcgtaccga tctgtttaaa cctagatatt gatagtctga
     5581 tcggtcaacg tataatcgag tcctagcttt tgcaaacatc tatcaagaga caggatcagc
     5641 aggaggcttt cgcatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     5701 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     5761 tcagttgggt gcgcgagtgg gttacatcga actggatctc aacagcggta agatccttga
     5821 gagttttcgc cccgaagaac gctttccaat gatgagcact tttaaagttc tgctatgtgg
     5881 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     5941 tcagaatgac ttggttgagt attcaccagt cacagaaaag catcttacgg atggcatgac
     6001 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     6061 tctgacaacg attggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     6121 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     6181 tgacaccacg atgcctgtag caatggcaac aaccttgcgt aaactattaa ctggcgaact
     6241 acttactcta gcttcccggc aacagttgat agactggatg gaggcggata aagttgcagg
     6301 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     6361 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     6421 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     6481 tgagataggt gcctcactga ttaagcattg gtaaccgatt ctaggtgcat tggcgcagaa
     6541 aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac atatgccaga ttcagcaacg
     6601 gatacggctt ccccaacttg cccacttcca tacgtgtcct ccttaccaga aatttatcct
     6661 taagatcccg aatcgtttaa actcgactct ggctctatcg aatctccgtc gtttcgagct
     6721 tacgcgaaca gccgtggcgc tcatttgctc gtcgggcatc gaatctcgtc agctatcgtc
     6781 agcttacctt tttggcagcg atcgcggctc ccgacatctt ggaccattag ctccacaggt
     6841 atcttcttcc ctctagtggt cataacagca gcttcagcta cctctcaatt caaaaaaccc
     6901 ctcaagaccc gtttagaggc cccaaggggt tatgctatca atcgttgcgt tacacacaca
     6961 aaaaaccaac acacatccat cttcgatgga tagcgatttt attatctaac tgctgatcga
     7021 gtgtagccag atctagtaat caattacggg gtcattagtt catagcccat atatggagtt
     7081 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     7141 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     7201 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     7261 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     7321 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     7381 taccatgctg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     7441 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     7501 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
     7561 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tcagatcttt
     7621 gtcgatccta ccatccactc gacacacccg ccagcggccg c
//
